Ovary-tissue transcriptional factors

ABSTRACT

Novel DNA constructs are provided which may be used as molecular probes or inserted into a plant host to provide for modification of transcription of a DNA sequence of interest in ovary tissue, particularly in very early fruit development. The DNA constructs comprise a transcriptional initiation regulatory region associated with gene expression in ovary tissue from immediately prior to anthesis through flower senescence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.07/998,158 filed Dec. 29, 1992 now U.S. Pat. No. 5,530,185 , which is acontinuation in part of U.S. application Ser. No. 07/554,195 filed Jul.17, 1990, now U.S. Pat. No. 5,175,095, which is a continuation-in-partof U.S. application Ser. No. 07/382,518, filed Jul. 19, 1989, nowabandoned which applications are incorporated herein by reference.

INTRODUCTION

1. Technical Field

This invention relates to methods of using in vitro constructed DNAtranscription or expression cassettes capable of directing ovary-tissuetranscription of a DNA sequence of interest in plants to produceovary-derived cells having an altered phenotype, and to methods ofproviding for or modifying existing color in various plant tissues orparts. The invention is exemplified by methods of using ovary tissuepromoters for altering the color phenotype of cotton fibers, and cottonfibers produced by the method.

2. Background

In general, genetic engineering techniques have been directed tomodifying the phenotype of individual prokaryotic and eukaryotic cells,especially in culture. Plant cells have proven more intransigent thanother eukaryotic cells, due not only to a lack of suitable vectorsystems but also as a result of the different goals involved. For manyapplications, it is desirable to be able to control gene expression at aparticular stage in the growth of a plant or in a particular plant part.For this purpose, regulatory sequences are required which afford thedesired initiation of transcription in the appropriate cell types and/orat the appropriate time in the plant's development without havingserious detrimental effects on plant development and productivity. It istherefore of interest to be able to isolate sequences which can be usedto provide the desired regulation of transcription in a plant cellduring the growing cycle of the host plant.

One aspect of this interest is the ability to change the phenotype ofparticular cell types, such as differentiated epidermal cells thatoriginate in ovary tissue, i.e. cotton fiber cells, so as to provide foraltered or improved aspects of the mature cell type. In order to effectthe desired phenotypic changes, transcription initiation regions capableof initiating transcription only in early ovary development are used.These transcription initiation regions are active prior to the onset ofpollination and are less active or inactive, before fruit enlargement,tissue maturation, or the like occur.

Relevant Literature

A class of fruit-specific promoters expressed at or during anthesisthrough fruit development, at least until the beginning of ripening, isdiscussed in European Application 88.906296.4, the disclosure of whichis hereby incorporated by reference. cDNA clones that are preferentiallyexpressed in cotton fiber have been isolated. One of the clones isolatedcorresponds to mRNA and protein that are highest during the late primarycell wall and early secondary cell wall synthesis stages. John Crow PNAS(1992) 89:5769-5773. cDNA clones from tomato displaying differentialexpression during fruit development have been isolated and characterized(Mansson et al., Mol. Gen. Genet. (1985) 200:356-361: Slater et al.,Plant Mol. Biol. (1985) 5:137-147). These studies have focused primarilyon mRNAs which accumulate during fruit ripening. One of the proteinsencoded-by the ripening-specific cDNAs has been identified aspolygalacturonase (Slater et al., Plant Mol. Biol. (1985) 5:137-147). AcDNA clone which encodes tomato polygalacturonase has been sequenced(Grierson et al., Nucleic Acids Research (1986) 14:8395-8603).Improvements in aspects of tomato fruit storage and handling throughtranscriptional manipulation of expression of the polygalacturonase genehave been reported (Sheehy et al., Proc. Natl. Acad. Sci. USA (1988)85:8805-8809; Smith et al., Nature (1988) 334:724-726).

Mature plastid mRNA for psbA (one of the components of photosystem II)reaches its highest level late in fruit development, whereas after theonset of ripening, plastid mRNAs for other components of photosystem Iand II decline to nondetectable levels in chromoplasts (Piechulla etal., Plant Molec. Biol. (1986) 7:367-376). Recently, cDNA clonesrepresenting genes apparently involved in tomato pollen (McCormick etal., Tomato Biotechnology (1987) Alan R. Liss, Inc., NY) and pistil(Gasser et al., Plant Cell (1989), 1:15-24) interactions have also beenisolated and characterized.

Other studies have focused on genes inducibly regulated, e.g. genesencoding serine proteinase inhibitors, which are expressed in responseto wounding in tomato (Graham et al., J. Biol. Chem. (1985)260:6555-6560: Graham et al., J. Biol. Chem. (1985) 260:6561-6554) andon mRNAs correlated with ethylene synthesis in ripening fruit and leavesafter wounding (Smith et al., Planta (1986) 168:94-100). Accumulation ofa metallocarboxypeptidase inhibitor protein has been reported in leavesof wounded potato plants (Graham et al., Biochem & BioPhys. Res Comm.(1981) 101:1164-1170).

Genes which are expressed preferentially in plant seed tissues, such asin embryos or seed coats, have also been reported. See, for example,European Patent Application 87306739.1 (published as 0 255 378 on Feb.3, 1988) and Kridl et al. (Seed Science Research (1991) 1:209-219).

Agrobacterium-mediated cotton transformation is described in Umbeck,U.S. Pat. Nos. 5,004,863 and 5,159,135 and cotton transformation byparticle bombardment is reported in WO 92/15675, published Sept. 17,1992. Transformation of Brassica has been described by Radke et al.(Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992)11:499-505.

Transformation of cultivated tomato is described by McCormick et al.,Plant Cell Reports (1986) 5:81-89 and Fillatti et al., Bio/Technology(1987) 5:726-730.

SUMMARY OF THE INVENTION

Novel DNA constructs and methods for their use are described which arecapable of directing transcription of a gene of interest in ovarytissue, particularly early in fruit development. The novel constructsinclude a vector comprising a transcriptional and translationalinitiation region obtainable from a gene expressed in ovary tissue andmethods of using constructs including the vector for altering fruitphenotype. The fruit may be edible or non-edible. The method includestransfecting a host plant cell of interest with a transcription orexpression cassette comprising a promoter which is active in ovary cellsprior to, and during, the pollination stage of the fruit, thengenerating a plant, which is grown to produce fruit having the desiredphenotype. Constructs and methods of the subject invention thus find usein modulation of endogenous fruit products, as well as production ofexogenous products and in modifying the phenotype of fruit and fruitproducts. The constructs also find use as molecular probes. Inparticular, constructs and methods for use in gene expression in cottonembryo tissues are considered herein. By these methods, novel cottonplants and cotton plant parts, such as modified cotton fibers, may beobtained.

Also provided in the instant application are constructs and methods ofuse relating to modification of color phenotype in plant tissues. Suchconstructs contain sequences for expression of genes involved in theproduction of colored compounds, such as melanin or indigo, and alsocontain sequences which provide for targeting of the gene products toparticular locations in the plant cell, such as plastid organelles, orvacuoles. Plastid targeting is of particular interest for expression ofgenes involved in aromatic amino acid biosynthesis pathways, whilevacuolar targeting is of particular interest where the precursorsrequired in synthesis of the pigment are present in vacuoles. Productionof melanin, for example, may be enhanced by vacuolar targeting in planttissues which accumulate tyrosine in vacuoles. Transcriptionalinitiation regions for expression of color-related genes will beselected on the basis of the tissue for which color modification isdesired.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence of cDNA clone (SEQ ID NO. 1) pZ130. Thesequences corresponding to the pZ7 cDNA clone are underlined.

FIG. 2 shows the sequence of the region of the Calgene Lambda 140genomic clone (SEQ ID NO. 1) that overlaps with the pZ130 cDNA clone(this region is underlined) and a partial sequence of regions 5′ and 3′to that region. The start of the pZ130 gene transcript is indicated bythe underlined, boldfaced “A” at position 2567. An intron in the genesequence is indicated by the lower case sequence from position 2702through position 2921. Sites for common restriction enzymes areindicated.

The symbols in the sequence have the following meaning:

A=adenosine; C=cytosine; G=guanine; T=thymidine or uracil; R=A or G; Y=Cor T or U; M=C or A; K=T or U or G; W=T or U or A; S=C or G; N=either C,T, A G or U; B=not A; D=not C; H=not G; V=not T or U.

FIG. 3 shows a restriction map of Calgene Lambda 140. B:BamHI; G:BglII;H:HindIII; R:EcoRI; S:SalI.

FIG. 4 shows a complete DNA sequence of cDNA clone (SEQ ID NO. 3) pZ70.The sequences corresponding to the pZ8 cDNA clone are underlined. Thestart and end of the mature protein encoded by the pZ70 gene are alsoindicated.

FIG. 5 shows a restriction map of Calgene Lambda 116. B:BamHI; G:BglII,H:HindIII; P:SphI; R:EcoRI; S:SalI; X:XbaI.

FIGS. 6A and 6B show the results of a Northern blot experimentillustrating a developmental time course of pZ7 (FIG. 6B) and pZ8 (FIG.6A) RNA accumulation. The stages of UC82B fruit development (flowers andovaries/fruit) are depicted in FIG. 6C Numbers 1 through 21 representdays post flower opening.

FIG. 7 shows a binary vector for plant transformation to express genesfor melanin synthesis.

FIG. 8 shows a linker region site map.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, novel constructs and methodsfor their use are described which may be used as molecular probes orinserted into a plant host to provide for transcription of a nucleotidesequence of interest in ovary cells as compared with other plant cells,generally preferentially in ovary cells to produce cells and plant partshaving an altered phenotype. Of particular interest is the period of atleast one to three days prior to anthesis through flower senescence.

The constructs include several forms, depending upon the intended use ofthe construct. Thus, the constructs include vectors, transcriptionalcassettes, expression cassettes and plasmids. The transcriptional andtranslational initiation region (also sometimes referred to as a“promoter,”), preferably comprises a transcriptional initiationregulatory region and a translational initiation regulatory region ofuntranslated 5′ sequences, “ribosome binding sites, ”responsible forbinding mRNA to ribosomes and translational initiation. It is preferredthat all of the transcriptional and translational functional elements ofthe initiation control region are derived from or obtainable from thesame gene. In some embodiments, the promoter will be modified by theaddition of sequences, such as enhancers, or deletions of nonessentialand/or undesired sequences. By “obtainable” is intended a promoterhaving a DNA sequence sufficiently similar to that of a native promoterto provide for the desired specificity of transcription of a DNAsequence of interest. It includes natural and synthetic sequences aswell as sequences which may be a combination of synthetic and naturalsequences.

The vectors typically comprise a nucleotide sequence of one or morenucleotides and a transcriptional initiation regulatory regionassociated with gene expression in ovary tissue. A transcriptionalcassette for transcription of a nucleotide sequence of interest in ovarytissue will include in the direction of transcription, an ovary tissuetranscriptional initiation region and optionally a translationalinitiation region, a DNA sequence of interest, and a transcriptional andoptionally translational termination region functional in a plant cell.When the cassette provides for the transcription and translation of aDNA sequence of interest it is considered an expression cassette. One ormore introns may be also be present.

Other sequences may also be present, including those encoding transitpeptides and secretory leader sequences as desired. The regulatoryregions are capable of directing transcription in ovary cells fromanthesis through flowering but direct little or no expression after theinitial changes which occur at the time surrounding pollination and/orfertilization; transcription from these regulatory regions is notdetectable at about three weeks after anthesis. Further, ovary-tissuetranscription initiation regions of this invention are typically notreadily detectable in other plant tissues. Transcription initiationregions from ovary tissue that are not ovary specific may find specialapplication. Especially preferred are transcription initiation regionswhich are not found at stages of fruit development other thanpre-anthesis through flowering. Transcription initiation regions capableof initiating transcription in other plant tissues and/or at otherstages of ovary development, in addition to the foregoing, areacceptable insofar as such regions provide a significant expressionlevel in ovary tissue at the defined periods of interest and do notnegatively interfere with the plant as a whole, and, in particular, donot interfere with the development of fruit and/or fruit-related parts.Also of interest are ovary tissue promoters and/or promoter elementswhich are capable of directing transcription in specific ovary tissuessuch as outer pericarp tissue, inner core tissues, integuments, and thelike.

Transcriptional initiation regions which are expressible in ovary tissueat or near maximal levels during the period of interest of thisinvention, generally the flowering period of plant reproductive cycles,are preferred. Of particular interest is the period of at least one tothree days prior to anthesis through flower senescence. Thetranscription level should be sufficient to provide an amount of RNAcapable of resulting in a modified fruit. The term “fruit” as usedherein refers to the mature organ formed as the result of thedevelopment of the ovary wall of a flower and any other closelyassociated parts. See Weirer, T. E., 1, ed., Botany A Introduction toPlant Biology (6th ed.) (John Wiley & Sons, 1982); Tootill & Backmore,The Facts on File Dictionary of Botany (Market Home Books Ltd., 1984).By “modified fruit” is meant fruit having a detectably differentphenotype from a nontransformed plant of the same species, for example,one not having the transcriptional cassette in question in its genome.

Of particular interest are transcriptional initiation regions associatedwith genes expressed in ovary tissue and which are capable of directingtranscription at least 24 hours prior to anthesis through flowersenescence. The term “anthesis” refers herein to the period associatedwith flower opening and flowering. The term “flower senescence” refersherein to the period associated with flower death, including the loss ofthe (flower) petals, etc. Abercrombie, M., et al., A Dictionary ofBiology (6th ed) (Penguin Books, 1973). Unopened flowers, or buds, areconsidered “pre-anthesis.”Anthesis begins with the opening of the flowerpetals, which represents asexually receptive portion of the reproductivecycle of the plant. Typically, flowering lasts approximately one week inthe tested UCB82 tomato variety. In a plant like cotton, flowering lastsapproximately two weeks and the fiber develops from the seed coattissue. It is preferred that the transcriptional initiation regions ofthis invention do not initiate transcription for a significant time orto a significant degree prior to plant flower budding. Ideally, thelevel of transcription will be high for at least approximately one tothree days and encompass the onset of anthesis (“pre-anthesis”).

It further is desired that the transcriptional initiation regions ofthis invention show a decreased level of transcriptional activity within1-3 days after the onset of anthesis which does not increase, andpreferably decreases over time. Fertilization of a tomato embryo sac, toproduce the zygote that forms the embryo plant, typically occurs 2-3days after flower opening. This coincides with a decrease in theactivity of a transcriptional initiation region of this invention. Thus,it is desired that the transcriptional activity of the promoter of thisinvention significantly decrease within about two days after the onsetof anthesis. Transcriptional initiation regions of this invention willbe capable of directing expression in ovary tissue at significantexpression levels during the preferred periods described above.

In some embodiments, it will be desired to selectively regulatetranscription in a particular ovary tissue or tissues. When used inconjunction with a 5′ untranslated sequence capable of initiatingtranslation, expression in defined ovary tissue, including ovaryinteguments (also known as “ovule epidermal cells”), core or pericarptissue, and the like, the transcriptional initiation region can direct adesired message encoded by a DNA sequence of interest in a particulartissue to more efficiently effect a desired phenotypic modification. Forexample, expression in ovary pericarp tissue, also known as the ovarywall and/or ovary core tissue, could result in useful modifications tothe edible portions of many fruits, including true berries such astomato, grape, blueberry, cranberry, currant, and eggplant; stone fruits(drupes), such as cherry, plum, apricot, peach, nectarine and avocado;and compound fruits (druplets), such as raspberry and blackberry. Inhesperidium (oranges, citrus), such expression cassettes are expected tobe expressed in the “juicy” portion of the fruit. In pepos, (such aswatermelon, cantaloupe, honeydew, cucumber, and squash) the equivalenttissue is most likely the inner edible portions. In other fruits, suchas legumes, the equivalent tissue is the seed pod.

The modification of analogous structures of non-edible fruit may also beof interest. Thus, of special interest are transcription initiationregions expressible in at least ovary outer pericarp tissue. Forexample, in cotton the analogous ovary structure is the burr of thecotton boll, in rapeseed it is the seed pod. In a like manner,regulating expression in ovary integuments and/or core tissue may resultin useful modifications to the analogous fruit and related structuresevolving there from, for example seed coat hairs, such as cotton fibers.Cotton fiber is a differentiated single epidermal cell of the outerintegument of the ovule. It has four distinct growth phases; initiation,elongation (primary cell wall synthesis), secondary cell wall synthesis,and maturation. Initiation of fiber development appears to be triggeredby hormones. The primary cell wall is laid down during the elongationphase, lasting up to 25 days postanthesis (DPA). Synthesis of thesecondary wall commences prior to the cessation of the elongation phaseand continues to approximately 40 DPA, forming a wall of almost purecellulose. In addition to ovary tissue promoters, transcriptionalinitiation regions from genes expressed preferentially in seed tissues,and in particular seed coat tissues, are also of interest forapplications where modification of cotton fiber cells is considered.

An example of a gene which is expressed at high levels in Brassica seedcoat cells is the EA9 gene described in EPA O255 378. The nucleic acidsequence of a portion of the EA9 cDNA is provided therein, and can beused to obtain corresponding sequences, including the promoter region.An additional seed gene which is expressed in seed embryo and seed coatcells is the Bce4 Brassica gene. The promoter region from this gene alsofinds use in the subject invention; this gene and the correspondingpromoter region are described in WO 91/13980, which was published Sept.19, 1991. Fiber specific proteins are developmentally regulated. Thus,transcriptional initiation regions from proteins expressed in fibercells are also of interest. An example of a developmentally regulatedfiber cell protein, is E6 (John and Crow Proc. Nat. Acad. Sci.(USA)(1992) 89:5769-5773). The E6 gene is most active in fiber, althoughlow levels of transcripts are found in leaf, ovule and flower.

To obtain a specifically derived transcriptional initiation region, thefollowing steps may be employed. Messenger RNA (mRNA) is isolated fromtissue of the desired developmental stage. This mRNA is then used toconstruct cDNA clones which correspond to the mRNA population both interms of primary DNA sequence of the clones and in terms of abundance ofdifferent clones in the population. mRNA is also isolated from tissue ofa different developmental stage in which the target gene should not beexpressed (alternate tissue). Radioactive cDNA from the desired tissueand from the alternate tissue is used to screen duplicate copies of thecDNA clones. The preliminary screen allows for classification of thecDNA clones as those which correspond to mRNAs which are abundant inboth tissues; those which correspond to mRNAs which are not abundant ineither tissue; those which correspond to mRNAs which are abundant in onetissue and relatively non-abundant in the other. Clones are thenselected which correspond to mRNAs that are abundant only in the desiredtissue and then these selected clones are further characterized.

Since the hybridization probe for the preliminary screen outlined aboveis total cDNA from a particular tissue, it hybridizes primarily to themost abundant sequences. In order to determine the actual level ofexpression, particularly in tissue where the mRNA is not as abundant,the cloned sequence is used as a hybridization probe to the total mRNApopulation(s) of the desired tissue(s) and various undesired tissue(s).This is most commonly done as a Northern blot which gives informationabout both the relative abundance of the mRNA in particular tissues andthe size of the mRNA transcript.

It is important to know whether the abundance of the mRNA is due totranscription from a single gene or whether it is the product oftranscription from a family of genes. This can be determined by probinga genomic Southern blot with the cDNA clone. Total genomic DNA isdigested with a variety of restriction enzymes and hybridized with theradioactive CDNA clone. From the pattern and intensity of thehybridization, one can distinguish between the possibilities that themRNA is encoded either by one or two genes or by a large family ofrelated genes. It can be difficult to determine which of severalcross-hybridizing genes encodes the abundantly expressed mRNA found inthe desired tissue. For example, tests indicate that pZ130 (see Example4) is a member of a small gene family however, the pZ7 probe is capableof distinguishing pZ130 from the remainder of the family members.

The cDNA obtained as described can be sequenced to determine the openreading frame (probable protein coding region) and the direction oftranscription so that a desired target DNA sequence later can beinserted at the correct site and in the correct orientation into atranscription cassette. Sequence information for the cDNA clone alsofacilitates characterization of corresponding genomic clones includingmapping and subcloning as described below. At the same time, a genomiclibrary can be screened for clones containing the complete gene sequenceincluding the control region flanking the transcribed sequences. Genomicclones generally contain large segments of DNA (approximately 10-20 kb)and can be mapped using restriction enzymes, then subcloned andpartially sequenced to determine which segments contain thedevelopmentally regulated gene.

Using the restriction enzyme map and sequence information, plasmids canbe designed and constructed which have the putative ovary gene or otherdesired promoter regions attached to genes which are to be expressed inovary and/or other desired tissue, particularly ovary-derived tissue.These hybrid constructions are tested for their pattern of expression intransformed, regenerated plants to be sure that the desired timingand/or tissue expression and/or the overall level of expression has beenmaintained successfully when the promoter is no longer associated withthe native open reading frame. Using the method described above, severaltranscriptional regulatory regions have been identified. One example isthe tomato-derived transcriptional initiation region which regulatesexpression of the sequence corresponding to the pZ130 cDNA clone.Sequences hybridizable to the pZ130 clone, for example, probe pZ7, showabundant mRNA, especially at the early stages of anthesis. The messageis expressed in ovary integument and ovary outer pericarp tissue and isnot expressed, or at least is not readily detectable, in other tissuesor at any other stage of fruit development. Thus, the pZ130transcriptional initiation region is considered ovary-specific forpurposes of this invention. FIG. 1 provides the DNA sequence of CDNAclone pZ130. The native function of the amino acid sequence encoded bythe structural gene comprising pZ130 is unknown.

Downstream from, and under the regulatory control of, the ovary tissuetranscriptional/translational initiation control region is a nucleotidesequence of interest which provides for modification of the phenotype ofstructures maturing from ovary tissue, such as fruit or fiber. Thenucleotide sequence may be any open reading frame encoding a polypeptideof interest, for example, an enzyme, or a sequence complementary to agenomic sequence, where the genomic sequence may be an open readingframe, an intron, a noncoding leader sequence, or any other sequencewhere the complementary sequence inhibits transcription, messenger RNAprocessing, for example, splicing, or translation. The nucleotidesequences of this invention may be synthetic, naturally derived, orcombinations thereof. Depending upon the nature of the DNA sequence ofinterest, it may be desirable to synthesize the sequence with plantpreferred codons. The plant preferred codons may be determined from thecodons of highest frequency in the proteins expressed in the largestamount in the particular plant species of interest. Phenotypicmodification can be achieved by modulating production either of anendogenous transcription or translation product, for example as to theamount, relative distribution, or the like, or an exogenoustranscription or translation product, for example to provide for a novelfunction or products in a transgenic host cell or tissue. Of particularinterest are DNA sequences encoding expression products associated withthe development of plant fruit, including genes involved in metabolismof cytokinins, auxins, ethylene, abscissic acid, and the like. Methodsand compositions for modulating cytokinin expression are described inU.S. Pat. No. 5,177,307, which disclosure is hereby incorporated byreference. Alternatively, various genes, from sources including othereukaryotic or prokaryotic cells, including bacteria, such as those fromAgrobacterium tumefaciens T-DNA auxin and cytokinin biosynthetic geneproducts, for example, and mammals, for example interferons, may beused.

Other phenotypic modifications include modification of the color ofplant parts developing from ovary integuments and/or core tissue, forexample seed coat hairs, such as cotton fibers. Of interest are genesinvolved in production of melanin and genes involved in the productionof indigo. Melanins are dark brown pigments found in animals, plants andmicroorganisms, any of which may serve as a source for sequences forinsertion into the constructs of the present invention. Specificexamples include the tyrosinase gene which can be cloned fromStreptomyces antibioticus. The ORF438 encoded protein in S. antibioticusalso is necessary for melanin production, and may provide a copper donorfunction. In addition, a tyrosinase gene can be isolated from anyorganism which makes melanin. The gene can be isolated from human hair,melanocytes or melanomas, cuttle fish and red roosters, among others.See, for example, EP Application No. 89118346.9 which discloses aprocess for producing melanins, their precursors and derivatives inmicroorganisms. Also, See, Bernan et al. Gene (1985) 37:101-110; anddella-Cioppa et al. Bio/Technology (1990) 8:634-638.

Indigo may be obtained by use of genes encoding a mono-oxygenase such asxylene oxygenase which oxidizes toluene and xylene to (methyl) benzylalcohol and also transforms indole to indigo. Cloning of the xyleneoxygenase gene and the nucleotide and amino acid sequences are describedin unexamined Japanese Patent Application Kokai:2-119777, published May7, 1990. A dioxygenase such as naphthalene dioxygenase which alsoconverts indole to indigo finds use; the naphthalene dioxygenase genenahA is described in Science (1983) 222:167. For cloning, nucleotidesequence in characterization of genes encoding naphthalene dioxygenaseof Pseudomonas putida. See, Kurkela et al. Gene (1988) 73:355-362. Atryptophanase gene sequence can be used in conjunction with an oxygenaseto increase the amount of indole available for conversion to indigo.Sources of tryptophanase gene sequences include E. coli (see, forexample, Deeley et al. (1982) J. Bacteriol. 151:942-951).

As demonstrated in the following examples, expression of ORF438 andtyrosinase genes from Streptomyces in transgenic tobacco plants using apZ7 promoter, and targeting the gene products to plastids by the actionof transit peptides resulted in phenotypic modification of tissues ovaryand meristem derived tissues, including modification of color inmeristematic regions and basal flower buds. A similar set of experimentsin which no plastid targeting sequences were used in conjunction withthe ORF438 and tyrosinase genes, no alteration of phenotype wasobserved. Presumably, the plants were not able to produce melanin due todeficiency of the required substrates in the plant cell cytosol. Plastidtargeting sequences (transit peptides) are available from a number ofplant nuclear-encoded plastid proteins, such as the small subunit (SSU)of ribulose bisphosphate carboxylase, plant fatty acid biosynthesisrelated genes including acyl carrier protein (ACP), stearoyl-ACPdesaturase, β-ketoacyl-ACP synthase and acyl-ACP thioesterase, or LHCPIIgenes. The encoding sequence for a transit peptide which provides fortransport to plastids may include all or a portion of the encodingsequence for a particular transit peptide, and may also contain portionsof the mature protein encoding sequence associated with a particulartransit peptide. There are numerous examples in the art of transitpeptides which may be used to deliver a target protein into a plastidorganelle. The particular transit peptide encoding sequence used in theinstant invention is not critical, as long as delivery to the plastid isobtained.

As an alternative to using transit peptides to target pigment synthesisproteins to plastid organelles, the desired constructs may be used totransform the plastid genome directly. In this instance, promoterscapable of providing for transcription of genes in plant plastids aredesired. Of particular interest is the use of a T7 promoter to providefor high levels of transcription. Since plastids do not contain anappropriate polymerase for transcription from the T7 promoter, T7polymerase may be expressed from a nuclear construct and targeted toplastids using transit peptides as described above. (See McBride et al.(1994) Proc. Nat. Acad. Sci. 91:7301-7305; see also U.S. Pat. No.5,925,806 entitled “Controlled Expression of Transgenic Constructs inPlant Plastids”, and U.S. Pat. No. 5,576,198 and PCT/US94/14574 filedDec. 12, 1994.) Tissue specific or developmentally regulated promotersmay be useful for expression of the T7 polymerase in order to limitexpression to the appropriate tissue or stage of development. Forexample, for flower color modification, the T7 polymerase may beexpressed from a petal specific promoter to limit effects to the desiredtissue.

Targeting of melanin synthesis genes to vacuoles is also of interest inplant tissues which accumulate the tyrosine substrate involved inmelanin synthesis in vacuoles. The protein signal for targeting tovacuoles may be provided from a plant gene which is normally transportedacross the rough endoplasmic reticulum, such as the 32 amino acidN-terminal region of the metallocarboxypeptidase inhibitor gene fromtomato (Martineau et al. (1991) Mol. Gen. Genet. 228:281-286). Inaddition to the signal sequence, vacuolar targeting constructs alsoencode a vacuolar localization signal (VLS) positioned at the carboxyterminus of the encoded protein. Appropriate signal sequences and VLSregions may be obtained from various other plant genes and may besimilarly used in the constructs of this invention. Numerous vacuolartargetting peptides are known to the art, as are reviewed in Chrispeelset al., Cell (1992) 68:613-616.

Thus, it is recognized that constructs of the instant invention whichprovide sequences encoding genes involved in color production andsequences which provide for targeting of the gene products toappropriate cellular locations have broad application to modification ofcolor in various plant tissues. Plant transcriptional initiation regionsfor use with these color modification constructs will depend upon theparticular plant tissue to be modified. For cotton fiber modification,for example, cotton fiber specific promoters or the pZ7 promoterdescribed herein may find use. Additional cotton fiber promoters whichmay find use in the methods of the instant application are described incopending US patent application to Pear et al., entitled “Cotton FiberTranscriptional Factors”, Ser. No. 08/480,178, filed on Jun. 7, 1995.For flower color modification, promoters from genes preferentiallyexpressed in flowers, and particularly in flower petals, are ofinterest. Examples of promoters useful for expression in flowers includechalcone synthase, as described in Holton et al. (1994) TIBTECH, Vol 12,pages 40-42(see also Napoli et al. (1990) Plant Cell, Vol 2, pages79-89; Lipphardt et al., (1988) EMBO, 7(13) pages 4027-4034; and Toguriet al., (1993) Plant Mol Biol, Vol 23, pages 933-946).

Also of interest are genes involved in production of colored pigments inplant tissues, such as the Maize A1 gene which encodes a dihydroflavonolreductase, an enzyme of the anthocyanin pigmentation pathway. In cellsthat express the A1 gene, dihydrokempferol is converted to 2-8alkylleucopelargonidin, which may be further metabolized to pelargonidinpigment by endogenous plant enzymes. Other anthocyanin or flavonoid typepigments may also be of interest for modification of cotton cell fibers,plant flowers or other plant tissues. For a review of plant flowercolor, see van Tunen et al. (in Plant Biotechnology Series, Volume 2(1990) Developmental Regulation of Plant Gene Expression, D. Griersoned.).

Although cotton fibers in commercially grown varieties are primarilywhite in color, other naturally occurring cotton varieties have brown orreddish-brown fibers. Also a cotton line containing green colored fibershas been identified. The existence of these colored cotton linessuggests that the precursors required for the anthocyanin pigmentpathways are present in cotton fibers cells, thus allowing further colorphenotype modifications.

For some applications, it is of interest to modify other aspects ofstructures developing from the ovary integument and related structures.For example, it is of interest to modify various aspects of cottonfibers, such as strength or texture of a fiber. Thus, the appropriategene may be inserted in the constructs of the invention, including genesfor PHB biosynthesis (see, Peoples et al. J. Biol. Chem. (1989)264:15298-15303 and Ibid. 15293-15397; Saxena, Plant Molecular Biology(1990) 15:673-683, which discloses cloning and sequencing of thecellulose synthase catalytic subunit gene; and Bowen et al. PNAS (1992)89:519-523) which discloses chitin synthase genes of Saccharomycescerevisiae and Candida albicans.

Transcriptional cassettes may be used when the transcription of ananti-sense sequence is desired. When the expression of a polypeptide isdesired, expression cassettes providing for transcription andtranslation of the DNA sequence of interest will be used. Variouschanges are of interest; these changes may include modulation (increaseor decrease) of formation of particular saccharides, hormones, enzymes,or other biological parameters. These also include modifying thecomposition of the final fruit or fiber, that is changing the ratioand/or amounts of water, solids, fiber or sugars. Other phenotypicproperties of interest for modification include response to stress,organisms, herbicides, brushing, growth regulators, and the like. Theseresults can be achieved by providing for reduction of expression of oneor more endogenous products, particularly an enzyme or cofactor, eitherby producing a transcription product which is complementary (anti-sense)to the transcription product of a native gene, so as to inhibit thematuration and/or expression of the transcription product, or byproviding for expression of a gene, either endogenous or exogenous, tobe associated with the development of a plant fruit.

The termination region which is employed in the expression cassette willbe primarily one of convenience, since the termination regions appear tobe relatively interchangeable. The termination region may be native withthe transcriptional initiation region, may be native with the DNAsequence of interest, may be derived from another source.

The termination region may be naturally occurring, or wholly orpartially synthetic. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. In some embodiments, it may bedesired to use the 3′ termination region native to the ovary tissuetranscription initiation region used in a particular construct.

As described herein, in some instances additional nucleotide sequenceswill be present in the constructs to provide for targeting of aparticular gene product to specific cellular locations. For example,where coding sequences for synthesis of aromatic colored pigments areused in a construct, particularly coding sequences for enzymes whichhave as their substrates aromatic compounds such tyrosine and indole, itis preferable to include sequences which provide for delivery of theenzyme into plastids, such as an SSU transit peptide sequence. Also, forsynthesis of pigments derived from tyrosine, such as melanin, targetingto the vacuole may provide for enhanced color modifications.

For melanin production, the tyrosinase and ORF438 genes fromStreptomyces antibioticus (Berman et al. (1985) 37:101-110) are providedin cotton fiber cells for expression from a pZ130 promoter. InStreptomyces, the ORF438 and tyrosinase proteins are expressed from thesame promoter region. For expression from constructs in a transgenicplant genome, the coding regions may be provided under the regulatorycontrol of separate promoter regions. The promoter regions may be thesame or different for the two genes. Alternatively, coordinateexpression of the two genes from a single plant promoter may be desired.Constructs for expression of the tyrosinase and ORF438 gene productsfrom pZ130 promoter regions are described in detail in the followingexamples. Additional promoters may also be desired, for example plantviral promoters, such as CaMV 35S, can be used for constitutiveexpression of one of the desired gene products, with the other geneproduct being expressed in cotton fiber tissues from the pZ130 promoter.In addition, the use of other plant promoters for expression of genes incotton fibers is also considered, such as the Brassica seed promotersand the E6 gene promoter discussed above. Similarly, other constitutivepromoters may also be useful in certain applications, for example themas, Mac or DoubleMac, promoters described in U.S. Pat. No. 5,106,739and by Comai et al., Plant Mol. Biol. (1990) 15:373-381). When plantscomprising multiple gene constructs are desired, for example plantsexpressing the melanin genes, ORF438 and tyrosinase, the plants may beobtained by co-transformation with both constructs, or by transformationwith individual constructs followed by plant breeding methods to obtainplants expressing both of the desired genes.

The various constructs normally will be joined to a marker for selectionin plant cells. Conveniently, the marker may be resistance to a biocide,particularly an antibiotic, such as kanamycin, G418, bleomycin,hygromycin, chloramphenicol, or the like. The particular marker employedwill be one which will allow for selection of transformed cells ascompared to cells lacking the DNA which has been introduced. Componentsof DNA constructs including transcription cassettes of this inventionmay be prepared from sequences which are native (endogenous) or foreign(exogenous) to the host. By foreign is intended that the sequence is notfound in the wild-type host into which the construct is introduced.Heterologous constructs will contain at least one region which is notnative to the gene from which the ovary tissue transcription initiationregion is derived.

In preparing the constructs, the various DNA fragments may bemanipulated, so as to provide for DNA sequences in the properorientation and, as appropriate, in proper reading frame for expression;adapters or linkers may be employed for joining the DNA fragments orother manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. In vitro mutagenesis, primer repair, restriction,annealing, resection, ligation, or the like may be employed, whereinsertions, deletions or substitutions, e.g. transitions andtransversions, may be involved. Conveniently, a vector or cassette mayinclude a multiple cloning site downstream from the ovary-relatedtranscription initiation region, so that the construct may be employedfor a variety of sequences in an efficient manner.

In carrying out the various steps, cloning is employed, so as to amplifythe amount of DNA and to allow for analyzing the DNA to ensure that theoperations have occurred in proper manner. By appropriate manipulations,such as restriction, chewing back or filling in overhangs to provideblunt ends, ligation of linkers, or the like, complementary ends of thefragments can be provided for joining and ligation. A wide variety ofcloning vectors are available, where the cloning vector includes areplication system functional in E. coli and a marker which allows forselection of the transformed cell. Illustrative vectors include pBR332,pUC series, M13mp series, pACYC184, etc. Thus, the sequence may beinserted into the vector at an appropriate restriction site(s), theresulting plasmid used to transform the E. coli host, the E. coli grownin an appropriate nutrient medium and the cells harvested and lysed andthe plasmid recovered. Analysis may involve sequence analysis,restriction analysis, electrophoresis, or the like. After eachmanipulation the DNA sequence to be used in the final construct may berestricted and joined to the next sequence. Each of the partialconstructs may be cloned in the same or different plasmids.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques include transfection with DNA employing A. tumefaciens or A.rhizogenes as the transfecting agent, protoplast fusion, injection,electroporation, particle acceleration, etc. For transformation withAgrobacterium, plasmids can be prepared in E. coli which contain DNAhomologous with the Ti-plasmid, particularly T-DNA. The plasmid may ormay not be capable of replication in Agrobacterium, that is, it may ormay not have a broad spectrum prokaryotic replication system such asdoes, for example, pRK290, depending in part upon whether thetranscription cassette is to be integrated into the Ti-plasmid or to beretained on an independent plasmid. The Agrobacterium host will containa plasmid having the vir genes necessary for transfer of the T-DNA tothe plant cell and may or may not have the complete T-DNA. At least theright border and frequently both the right and left borders of the T-DNAof the Ti- or Ri-plasmids will be joined as flanking regions to thetranscription construct. The use of T-DNA for transformation of plantcells has received extensive study and is amply described in EPA Ser.No. 120,516, Hoekema, In: The Binary Plant Vector SystemOffset-drukkerij Kanters B.V., Alblasserdam, 1985, Chapter V, Knauf, etal., Genetic Analysis of Host Range Expression by Agrobacterium, In:Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed.,Springer-Verlag, NY, 1983, p. 245, and An, et al., EMBO J. (1985)4:277-284.

For infection, particle acceleration and electroporation, a disarmedTi-plasmid lacking particularly the tumor genes found in the T-DNAregion may be introduced into the plant cell. By means of a helperplasmid, the construct may be transferred to the A. tumefaciens and theresulting transfected organism used for transfecting a plant cell;explants may be cultivated with transformed A. tumefaciens or A.rhizogenes to allow for transfer of the transcription cassette to theplant cells. Alternatively, to enhance integration into the plantgenome, terminal repeats of transposons may be used as borders inconjunction with a transposase. In this situation, expression of thetransposase should be inducible, so that once the transcriptionconstruct is integrated into the genome, it should be relatively stablyintegrated. Transgenic plant cells are then placed in an appropriateselective medium for selection of transgenic cells which are then grownto callus, shoots grown and plantlets generated from the shoot bygrowing in rooting medium.

To confirm the presence of the transgenes in transgenic cells andplants, a Southern blot analysis can be performed using methods known tothose skilled in the art. Expression products of the transgenes can bedetected in any of a variety of ways, depending upon the nature of theproduct, and include immune assay, enzyme assay or visual inspection,for example to detect pigment formation in the appropriate plant part orcells. Once transgenic plants have been obtained, they may be grown toproduce fruit having the desired phenotype. The fruit or fruit parts,such as cotton fibers may be harvested, and/or the seed collected. Theseed may serve as a source for growing additional plants having thedesired characteristics. The terms transgenic plants and transgeniccells include plants and cells derived from either transgenic plants ortransgenic cells.

The various sequences provided herein may be used as molecular probesfor the isolation of other sequences which may be useful in the presentinvention, for example, to obtain related transcriptional initiationregions from the same or different plant sources. Relatedtranscriptional initiation regions obtainable from the sequencesprovided in this invention will show at least about 60% homology, andmore preferred regions will demonstrate an even greater percentage ofhomology with the probes. Of particular importance is the ability toobtain related transcription initiation control regions having thetiming and tissue parameters described herein. For example, using theprobe pZ130, at least 7 additional clones, have been identified, but notfurther characterized. Thus, by employing the techniques described inthis application, and other techniques known in the art (such asManiatis, et al., Molecular Cloning, —A Laboratory Manual (Cold SpringHarbor, N.Y.) 1982), other transcription initiation regions capable ofdirecting ovary tissue transcription as described in this invention maybe determined. The constructs can also be used in conjunction with plantregeneration systems to obtain plant cells and plants; the constructsmay also be used to modify the phenotype of a fruit and fruits producedthereby.

For flower color modification, transformation of various flowering plantspecies is desired, including transformation of carnations, roses,gerba, lillies, orchids, petunias and chrysanthemums. For cottonapplications, various varieties and lines of cotton may find use in thedescribed methods. Cultivated cotton species include Gossypium hirsutumand G. babadense (extra-long stable, or Pima cotton), which evolved inthe New World, and the Old World crops G. herbaceum and G. arboreum. Thefollowing examples are offered by way of illustration and not bylimitation.

EXPERIMENTAL

The following deposits have been made at the American Type CultureCollection (ATCC) (12301 Parklawn Drive, Rockville, Md. 20852).Bacteriophage Calgene Lambda 116 and Calgene Lambda 140, each containinga transcription initiation region of this invention, were deposited onJul. 13, 1989 and were given accession numbers 40632 and 40631,respectively.

EXAMPLE 1 Construction of Pre-Anthesis Tomato Ovary cDNA Banks andScreening for Ovary-Specific Clones cDNA Library Preparation

Tomato plants (Lycopersicon esculentum cv UC82B) were grown undergreenhouse conditions. Poly(A)+RNA was isolated as described by Manssonet al., Mol. Gen. Genet. (1985) 200:356-361. The synthesis of cDNA frompoly(A)+RNA, prepared from ovaries of unopened tomato flowers(pre-anthesis stage), was carried out using the BRL cDNA Cloning Kitfollowing the manufacturer's instructions (BRL; Bethesda, Md.). Additionof restriction endonuclease EcoRI linkers (1078, New England Biolabs;Beverly, Mass.) to the resulting double-stranded cDNA was accomplishedby using the procedures described in Chapter 2 of DNA Cloning Vol. I: APractical Approach, Glover, ed., (BRL Press, Oxford 1985). Cloning thecDNA into the EcoRI site of the phage Lambda ZAP (Stratagene; La Jolla,Calif.) and packaging the resulting recombinant phage (using GigaPackGold, Stratagene) was carried out as described in the respectivecommercial protocols.

Two cDNA libraries were prepared as described above from the samepre-anthesis stage mRNA. For the second library, which containedsignificantly longer cDNA than the first, the poly(A)+RNA sample was runthrough an RNA spin column (Boehringer Mannheim Biochemicals;Indianapolis, Ind.), following the manufacturer's directions, prior tothe cloning procedures.

cDNA Library Screening

The first cDNA library was screened by differential hybridization using³²P-labeled CDNA probes made from pre-anthesis mRNA, leaf mRNA and youngseedling mRNA. Clones were selected based on hybridization to onlypre-anthesis mRNA. The cDNAs corresponding to the selected Lambda ZAP(Stratagene) clones were excised from the phage vector and propagated asplasmids (following the manufacturer's instructions).

From an initial screen of 1000 cDNAs, 30 selected clones falling intofive classes based on the sequences of their cDNA inserts were isolated.Two clones, clones pZ7 and pZ8, were selected for further study. The DNAsequences of pZ7 and pZ8 are shown as the underlined portions of FIGS. 1and 4, respectively.

Several thousand recombinant clones from the second cDNA library werescreened by plaque hybridization (as described in the Stratagene CloningKit Instruction Manual) with a mixture of radiolabeled DNA probes.Screening of approximately three thousand recombinant clones from thesecond library with the pZ7 and pZ8 DNA probes yielded selection offourteen clones which had intense hybridization signals. The clonesselected were excised from the phage vector and propagated as plasmids.DNA was isolated from each clone, cut with the restriction endonucleaseEcoRI, then electrophoresed through a 0.7% agarose gel. Duplicate blothybridizations were performed as described in Maniatis et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 1982) withradiolabeled probes representing the genes of interest (pZ7 and pZ8).Seven clones which hybridized to pZ7 and three clones which hybridizedto pZ8 were selected. The longest of these for each probe, pZ130(pZ7-hybridizing) and pZ70 (pZ8-hybridizing), were characterized furtherand used in additional experiments.

EXAMPLE 2 Analysis of cDNA Clones Northern Analysis

Tissue-specificity of the cDNA clones was demonstrated as follows: RNAwas isolated from 1, 2, 3, 4, 5, 6, 7, 10, 14, 17 and 21 daypost-anthesis, anthesis and pre-anthesis stage tomato ovaries, tomatoleaves and unorganized tomato callus using the method of Ecker andDavis, Proc. Natl. Acad. Sci. USA, 84:5203 (1987) with the followingmodifications. After the first precipitation of the nucleic acid, thepellets were resuspended in 2 ml of diethylpyrocarbonate (DEP)treatedwater on ice. The solutions were brought to 1 mM MgC12 and ¼ volume of 8M LiCl was added. The samples were mixed well and stored at 4° C.overnight. The samples were then centrifuged at 8,000 RPM for 20 min. at4° C. The pellets were dried, resuspended in DEP-treated water on ice asbefore and ethanol-precipitated once more. The RNAs were electrophoresedon formaldehyde/agarose gels according to the method described byFourney et al., Focus (1988) 10:5-7, immobilized on Nytran membranes(Schleicher & Schuell; Keene, N. H.) and hybridized with ³²P-labeledprobes.

Based upon the Northern analysis with a ³²P-labeled pZ7 EcoRI insert DNAor a pZ8 EcoRI insert DNA, it is clear that both of these genes are mosthighly expressed at anthesis in tomato variety UC82B and somewhat lesshighly expressed prior to and a day following the opening of the flower.FIG. 6 shows tomato flowers at various stages of development andimmediately below, a representative ovary dissected from a flower at thesame stage of development. As seen in FIG. 6, by two days after theonset of anthesis, the expression of both genes had dropped offdramatically. The size of the mRNA species hybridizing to the pZ7 probewas approximately 800 nt and to the pZ8 probe approximately 500 nt.

From two days post-anthesis, pZ8 RNA accumulation was apparentlymaintained at a relatively low level while pZ7 RNA accumulationcontinued to drop off steadily until, by three weeks post-anthesis, itwas undetectable by this analysis. pZ8 RNA accumulation was notdetectable by the method described above in RNA samples isolated fromtomato fruit older than the immature green stage of fruit ripening. NoRNA hybridizing to pZ7 or pZ8 was found in callus tissue; no RNAhybridizing to pZ7 was found in leaf tissue; on longer exposures abarely detectable hybridization signal for pZ8 was seen in leaf RNA.

Expression Level

Message abundance corresponding to the cDNA probes was determined bycomparing the hybridization intensity of a known amount of RNAsynthesized in vitro from the clones (using T7 or T3 RNA polymerase inthe Riboprobe System (Promega)) to RNA from anthesis stage and threeweek old tomato ovaries. This analysis indicated that pZ7 and pZ8 cDNAsrepresent abundant RNA classes in anthesis-stage tomato ovaries, beingapproximately 5% and 2% of the message, respectively.

Cellular Specificity

The cellular specificity of the cDNA probes may be demonstrated usingthe technique of in situ hybridization. Pre-anthesis stage UC82B tomatoovaries were fixed overnight in a 4% paraformaldehyde, phosphatebuffered saline (PBS), 5 MM MgCl₂ solution, pH 7.4 (PBS is 10 mMphosphate buffer, pH 7.4, 150 mM NaCl) (Singer et al., Biotechniques(1986) 4:230-250). After fixation, the tissue was passed through agraded tertiary butyl alcohol (TBA) series, starting at 50% alcohol,infiltrated with Paraplast and cast into paraffin blocks for sectioning(Berlyn and Miksche, Botanical Microtechnique and Cytochemistry, (1976)Iowa). Embedded ovaries were transversely cut, 8 μm thick sections, on aReichert Histostat rotary microtome. Paraffin ribbons holding 5-7 ovarysections were affixed to gelatin-chrom alum subbed slides (Berlyn andMiksche (1976) supra) and held in a dust-free box until in situhybridizations were performed. Slides ready to be hybridized weredeparaffinized in xylene and rehydrated by passing through an ethanolhydration series as described in Singer et al., supra (1986).

A 2× hybridization mix was made consisting of 100 μl 20×SSC, 20 μl 10%BSA, 100 μl 750 mM DTT, 200 μl 50% dextran sulfate, 50 μl RNasin, and 30μl sterile water. Sense and antisense ³⁵S-RNA probes were generated fromcDNAs of interest using T3 and T7 RNA polymerases in vitro transcription(Riboprobe Promega Biotec or Stratagene) reactions following themanufacturer's protocol. 2.5 μl tRNA (20 mg/ml), 2.5 μl salmon sperm DNA(10 mg per ml) and 4×10⁶ cpm/ probe were dried down using a lyophilizer.This mix was then resuspended in 25 μl 90% formamide containing 25 μl 2×hybridization mix per slide. 40 μl of this hybridization mix was placedon each slide. A cover slip was placed over the sections and edgessealed with rubber cement. Slides were placed in slide holders inside aglass slide box, covered, and placed in a 37° C. dry oven overnight tohybridize. Posthybridization treatments were as described in Singer etal., (1986), supra.

Autoradiography was performed as described in KODAK Materials for LightMicroscope (KODAK (1986); Rochester, N.Y.) using liquid emulsion NTB-3.Slides are left to expose in a light-tight box for approximately twoweeks. After developing the autoradiographic slides, sections werestained in 0.05% toluidine blue and then dehydrated through a gradedalcohol series; xylene:100% ethanol, 1:1, followed by 2 changes of 100%xylene, five minutes in each solution. Coverslips were mounted withCytoseal (VWR; San Francisco, Calif.) and left on a slide warmer untildry (45-50° C., 1-2 days). Autoradiographic slides were then ready formicroscopic examination.

When pre-anthesis tomato ovaries were hybridized to sense and antisense35S-pZ7 RNA, the antisense transcripts hybridized specifically to theouter pericarp region of the ovary and to the outer region of the ovules(the integuments). The sense transcripts (negative control) showed nohybridization. When pre-anthesis tomato ovaries were hybridized to senseand antisense 35S-pZ8 RNA, the antisense transcript hybridizedspecifically to the inner core region of the ovary and to the outerregion of the ovules. The sense transcripts showed no hybridization.

In summary, the mRNA transcripts encoded by the genes corresponding topZ7 and pZ8 were abundantly expressed during a very specific stage oftomato fruit development, primarily at anthesis and at a day prior toand after the opening of the flower. The transcripts additionally wereexpressed in a specific subset of tomato ovary cell types during thatstage of development particularly in the integuments (pZ7 and pZ8) aswell as the ovarian outer pericarp (pZ7) and inner core region (pZ8).

EXAMPLE 3 Sequencing of DZ130 and pZ70 cDNA Clones

The complete DNA sequences of the cDNA pZ130 and pZ70 clones weredetermined using the Sanger et al. (1971) dideoxy technique. The DNAsequences of both pZ130 and pZ70 were translated in three frames. Thesequences, including the longest open reading frame for each, are shownin FIG. 1 (pZ130) and FIG. 4 (pZ70).

EXAMPLE 4 Analysis of Gene Family

Southern analysis was performed as described by Maniatis et al., supra,(1982). Total tomato DNA from cultivar UC82B was digested with BamHI,EcoRI and HindIII , separated by agarose gel electrophoresis andtransferred to nitrocellulose. Southern hybridization was performedusing ³²P-labeled probes produced by random priming of pZ130 or pZ70. Asimple hybridization pattern indicated that the genes encoding pZ130 andpZ70 are present in a few or perhaps only one copy in the tomato genome.

Additional analysis, using a pZ130 hybridization probe to hybridize totomato genomic DNA digested with the restriction endonuclease BglII,indicated that this gene is actually a member of a small (approximately5-7 member) family of genes. The original pZ7 CDNA clone, consisting ofsequences restricted to the 3′ untranslated region of the longer pZ130clone, however, hybridizes intensely only to one band and perhapsfaintly to a second band based on Southern analysis using BglII digestedtomato genomic DNA.

EXAMPLE 5 Preparation of Genomic Clones DZ130 and pZ70

Two genomic clones, one representing each of cDNA clones pZ130 and pZ70,were obtained as follows. A genomic library constructed from DNA of thetomato cultivar UC82B, partially digested with the restrictionendonuclease Sau3A, was established in the lambda phage vector,lambda-FIX according to the manufacturer's instructions (Stratagene; LaJolla, Calif.). This library was screened using ³²P-labeled pZ130 andpZ70 as probes. A genomic clone containing approximately 14.5 kb ofsequence from the tomato genome which hybridized to pZ70 was isolated.The region which hybridizes to the pZ70 probe was found within theapproximately 2 kb XbaI-HindlII restriction fragment of Calgene Lambda116 (See FIG. 5). A second genomic clone, containing approximately 13 kbof sequence from the tomato genome and hybridizing to pZ130 (and pZ7)was isolated. The region which hybridized to the pZ130 probe was foundwithin the larger EcoRI-HindIII restriction fragment of Calgene Lambda140 (See FIG. 3).

Preparation of pCGN2015

pCGN2015 was prepared by digesting pCGN565 with HhaI, blunting with mungbean nuclease, and inserting the resulting fragment into an EcoRVdigested BluescriptKSM13-(Stratagene) vector to create pCGN2008.pCGN2008 was digested with EcoRI and HindIII, blunted with Klenow, andthe 1156 bp chloramphenicol fragment isolated. BluescriptKSM13+(Stratagene) was digested with DraI and the 2273 bp fragment isolatedand ligated with the pCGN2008 chloramphenicol fragment creatingpCGN2015.

Preparation of pCGN2901/pCGN2902

pCGN2901 contains the region surrounding the pZ7-hybridizing region ofthe pZ130 genomic clone, including approximately 1.8 kb in the 5′direction and approximately 4 kb in the 3′-direction. To preparepCGN2901, Calgene Lambda 140 was digested with SalI and the resultingfragment which contains the pZ7-hybridizing region was inserted intopCGN2015, at the pCGN2015 unique SalI site, to create pCGN2901.

pCGN2902 contains the other SalI fragment (non-pZ7-hybridizing) of thepZ130 genome derived from SalI digestion of Calgene Lambda 140, also putinto a pCGN2015 construct.

EXAMPLE 6 Preparation of a pZ130 Expression Construct

Plasmid DNA isolated from pCGN2901 was digested to completion with NcoIand then treated with exonuclease isolated from mung bean (Promega,Madison, Wis.) to eliminate single-stranded DNA sequences including theATG sequence making up a portion of the NcoI recognition sequence. Thesample was then digested to completion with SacI. The resulting 1.8 kb(approximate) 5′ SacI to NcoI fragment was then inserted into apUC-derived ampicillin-resistant plasmid, pCGP261 (described below),that had been prepared as follows. pCGP261 was digested to completionwith XbaI, the single-stranded DNA sequences were filled in by treatmentwith the Klenow fragment of DNA polymerase I, and the pCGP261 DNAredigested with SacI. The resulting expression construct contained, inthe 5′ to 3′ direction of transcription, an ovary tissue promoterderived from Lambda 140, a tmr gene and tmr 3′-transcriptionaltermination region.

The plasmid pCGP261 contains the sequences from position 8,762 through9,836 from the Agrobacterium tumefaciens octopine Ti plasmid pTil5955(as sequenced by Barker et al., Plant Molec. Biol. (1983) 2:335-350).This region contains the entire coding region for the genetic locusdesignated tmr which encodes isopentenyltransferase (Akiyoshi et al.,PNAS (1984) 81:4776-4780), 8 bp 5′ of the translation initiation ATGcodon and 341 bp of sequences 3′ to the translation stop TAG codon.

Plasmid pCGP261 was created as follows. Plasmid pCGN1278 (described inco-pending application U.S. Ser. No. 07/382,176, filed Jul. 19, 1989,which is hereby incorporated in its entirety by reference) was digestedwith XbaI and EcoRI. The single-stranded DNA sequences produced werefilled in by treatment with the Klenow fragment of DNA polymerase I. TheXbaI to EcoRI fragment containing the tmr gene was then ligated into thevector ml3 Bluescript minus (Stratagene Inc., La Jolla, Calif.) at theSmaI site, resulting in plasmid pCGP259. All of the region foundupstream of the ATG translation initiation codon and some of the tmrgene coding region was eliminated by digesting pCGP259 with BspMI andBstXI. The resulting coding region and 8 bp of the sequence originallyfound upstream of the first ATG codon was re-introduced into the plasmidand an XbaI site introduced into the plasmid via a syntheticoligonucleotide comprising the following sequence (SEQ ID NO. 4): 5′AATTAGATGCAGGTCCATAAGTTTTTTCTAGACGCG 3′. The resulting plasmid ispCGP261. An XbaI to KpnI fragment of pCGP261 containing the pZ130 gene5′ and tmr gene coding and 3′ region construct was then inserted into abinary cassette such as pCGN1557 and transgenic plants prepared. (Seeco-pending application U.S. Ser. No., 07/382,176 described above).

EXAMPLE 7 Preparation of pZ130 Promoter Cassette

The pZ130 cassette contains 1.8 kb (pCGN2909) or 5 kb (pCGN2928) of DNA5′ of the translational start site and the 3′ region (from the TAA stopcodon to a site 1.2 kb downstream) of the pZ130 gene. The pZ130cassettes were constructed as follows.

Transcriptional Initiation Region

Plasmid DNA isolated from pCGN2901 (see above) was digested tocompletion with NcoI and then treated with exonuclease isolated frommung bean (Promega, Madison, Wis.) to eliminate single-stranded DNAsequences, including the ATG sequence making up a portion of the NcoIrecognition sequence. The sample was then digested to completion withSacI. The resulting 1.8 kb 5′ SacI to NcoI fragment was then insertedinto pCGN2015 (described above) to create pCGN2904.

In order to eliminate redundant restriction enzyme sites and makesubsequent cloning easier, plasmid DNA isolated from pCGN2904 wasdigested to completion with SalI and EcoRI and the resulting 1.8 kbfragment, containing the pZ130 5′ sequences, inserted into pBluescriptII(Stratagene; La Jolla, Calif.) to create pCGN2907.

Transcriptional and Translational Termination Region

Plasmid DNA isolated from pCGN2901 was digested to completion with EcoRIand BamHI. The resulting 0.72 kb EcoRI to BamHI fragment locateddownstream (3′) from the pZ130 coding region was inserted into pCGN2907creating pCGN2908.

The insertion of the 0.5 kb (approximately) DNA sequence, including thepZ130 gene TAA stop codon and those sequences between the stop codon andthe EcoRI site downstream (3′) and the addition of unique restrictionsites to facilitate insertion of foreign genes, was accomplished asfollows.

A polylinker/“primer” comprising the sequence (SEQ ID NO. 5)5′GTTCCTGCAGCATGCCCGGGATCGATAATAATTAAGTGAGGC-3′ was synthesized tocreate a polylinker with the following sites: PstI-SphI-SmaI-ClaI and toinclude the pZ130 gene TAA stop codon and the following (3′) 13 basepairs of the pZ130 gene 3′ region sequence. Another oligonucleotidecomprising the sequence (SEQ ID NO. 6) 5′- CAAGAATTCATAATATTATATATAC-3′was synthesized to create a “primer” with an EcoRI restriction site and16 base pairs of the pZ130 gene 3′ region immediately adjacent to theEcoRI site located approximately 0.5 kb 3′ of the pZ130 gene TAA stopcodon.

These synthetic oligonucleotides were used in a polymerase chainreaction (PCR) in which plasmid DNA isolated from pCGN2901 was used asthe substrate in a thermal cycler (Perkin-Elmer/Cetus, Norwalk, Conn.)as per the manufacturer's instructions. The resulting 0.5 kb DNA productwas digested to completion with PstI and EcoRI and the resulting 0.5 kbDNA fragment inserted into pCGN2908 to create pCGN2909. The complete DNAsequence of the 0.5 kb region from the PstI site to the EcoRI site wasdetermined using the Sanger et al. (1971) dideoxy technique to verifythat no mistakes in the sequence had occurred between theoligonucleotide primers during the PCR reaction.

The pZ130 cassette, pCGN2909, thus comprises the 5′ pZ130 DNA sequencesfrom the SalI site at position 808 to position 2636 (see FIG. 2), uniquePstI, SphI and SmaI sites which can be conveniently used to insertgenes, and the 3′ pZ130 DNA sequences from the TAA stop codon atposition 3173 (FIG. 2) through the BamHI site at position 4380.

EXAMPLE 8 Preparation and Analysis of Test Constructs

A β-glucuronidase (GUS) reporter gene was used to evaluate theexpression and tissue specificity of the pZ130-GUS constructions. GUS isa useful reporter gene in plant systems because it produces a highlystable enzyme, there is little or no background (endogenous) enzymeactivity in plant tissues, and the enzyme is easily assayed usingfluorescent or spectrophotometric substrates. (See, for example,Jefferson, Plant Mol. Rep. (1987) 5:387-405.) Histochemical stains forGUS enzyme activity are also available which can be used to analyze thepattern of enzyme accumulation in transgenic plants. Jefferson (1987),supra.

A pZ130 cassette, pCGN2928, was prepared by inserting the 3.2 KpnI toSalI fragment of pCGN2059 into the KpnI and SalI sites of pCGN2909.pCGN2059 was prepared by inserting the 3.2 SalI to BglII fragment ofpCGN2902 into M13mp19. pCGN2928 is thus identical to pCGN2909 exceptthat it includes an additional approximately 3.2 kb of pZ130 DNAsequence upstream of the SalI site located at position 808 of FIG. 2.

Preparation of Test Constructs pCGN2917 and pCGN2918

These constructs contain 1.8 kb of pZ130 5′ sequence, the GUS genecoding region and 1.2 kb of pZ130 3′ sequence. pCGN2917 and pCGN2918differ from each other only in the orientation of the pZ130/GUSconstruction with respect to the other elements of the binary vectorplasmid for example, the 35S promoter from CaMV.

The constructs were made by inserting the PstI fragment of pRAJ250(Jefferson (1987) supra), or any other plasmid construct having the PstIfragment containing the GUS coding region, into the PstI site ofpCGN2909. The resulting plasmid, having the GUS gene in the senseorientation with respect to the pZ130 gene promoter region, was namedpCGN2914. The pZ130/GUS construction was excised as an XbaI to KpnIfragment and cloned into the binary vectors pCGN1557 and pCGN1558 tomake pCGN2917 and pCGN2918, respectively. pCGN1557 and pCGN1558 aredescribed in McBride and Summerfelt, Plant Mol. Bio. (1990) 14:269-296.

Preparation of Test Construct pCGN2926

This construct contains S kb of pZ130 5′ sequence, the GUS gene codingregion and 1.2 kb of pZ130 3′ sequence. It was made by inserting the 3.2kb KpnI to SalI fragment of pCGN2059 into the KpnI and SalI sites ofpCGN2914. The resulting plasmid was named pCGN2923. The pZ130/GUS/pZ130construction was then excised from pCGN2923 as an XbaI to KpnI fragmentand cloned into the binary vector pCGN1557 resulting in pCGN2926.

Analysis of GUS Enzyme Activity

β-glucuronidase activity of transformants was measured using4-methyl-umbelliferyl glucuronide as a substrate, as outlined inJefferson (1987) supra GUS enzyme activity was easily detected in theovaries of the transformed plants and quantitatively was quite high incomparison with the activity background observed in ovaries isolatedfrom nontransformed tomato plants and from leaves of transformed plants.Interestingly, upon comparison of the pCGN2917 and pCGN2918transformants, it was found that proximity to a 35S CaMV enhancer region(pCGN1558) may reduce, or eliminate, ovary-tissue specificity.

EXAMPLE 9 PZ-7 Cotton Transformation Explant Preparation

Coker 315 seeds were surface disinfected by placing in 50% Clorox (2.5%sodium hypochlorite solution) for 20 minutes and rinsing 3 times insterile distilled water. Following surface sterilization, seeds weregerminated in 25×150 sterile tubes containing 25 mls ½×MS salts: ½×B5vitamins: 1.5% glucose: 0.3% gelrite. Seedlings were germinated in thedark at 28° C. for 7 days. On the seventh day seedlings were placed inthe light at 28±2° C.

Cocultivation and Plant Regeneration

Single colonies of A. tumefaciens strain 2760 containing binary plasmidspCGN2917 and pCGN2926 were transferred to 5 ml of MG/L broth and grownovernight at 30° C. Bacteria cultures were diluted to 1×10⁸ cells/mlwith MG/L just prior to cocultivation. Hypocotyls were excised fromeight day old seedlings, cut into 0.5-0.7 cm sections and placed ontotobacco feeder plates (Horsch et al. 1985). Feeder plates were preparedone day before use by plating 1.0 ml tobacco suspension culture onto apetri plate containing Callus Initiation Medium CIM without antibiotics(MS salts: B5 vitamins: 3% glucose: 0.1 mg/L 2,4-D: 0.1 mg/L kinetin:0.3% gelrite, pH adjusted to 5.8 prior to autoclaving). A sterile filterpaper disc (Whatman #1) was placed on top of the feeder cells prior touse. After all sections were prepared, each section was dipped into anA. tumefaciens culture, blotted on sterile paper towels and returned tothe tobacco feeder plates.

Following two days of cocultivation on the feeder plates, hypocotylsections were placed on fresh Callus Initiation Medium containing 75mg/L kanamycin and 500 mg/L carbenicillin. Tissue was incubated at 28±2°C., 30 uE 16:8 light:dark period for 4 weeks. At four weeks the entireexplant was transferred to fresh callus initiation medium containingantibiotics. After two weeks on the second pass, the callus was removedfrom the explants and split between Callus Initiation Medium andRegeneration Medium (MS salts: 40 mM KNO₃: 10 mM NH₄Cl: B5 vitamins: 3%glucose: 0.3% gelrite: 400 mg/L carb: 75 mg/L kanamycin).

Embryogenic callus was identified 2-6 months following initiation andwas subcultured onto fresh regeneration medium. Embryos were selectedfor germination, placed in static liquid Embryo Pulsing Medium (Stewartand Hsu, medium: 0.01 mg/1 NAA: 0.01 mg/L kinetin: 0.2 mg/L GA3) andincubated overnight at 30° C. The embryos were blotted on paper towelsand placed into Magenta boxes containing 40 mls of Stewart and Hsumedium solidified with Gelrite. Germinating embryos were maintained at28±2° C. 50 uE m⁻²s⁻¹ 16:8 photoperiod. Rooted plantlets weretransferred to soil and established in the greenhouse.

Cotton growth conditions in growth chambers are as follows: 16 hourphotoperiod, temperature of approximately 80-85°, light intensity ofapproximately 500 μEinsteins. Cotton growth conditions in greenhousesare as follows: 14-16 hour photoperiod with light intensity of at least400 μEinsteins, day temperature 90-95° F., night temperature 70-75° F.,relative humidity to approximately 80%.

Plant Analysis

Flowers from greenhouse grown Tl plants were tagged at anthesis in thegreenhouse. Squares (cotton flower buds), flowers, bolls etc. wereharvested from these plants at various stages of development and assayedfor GUS activity. GUS fluorometric and histochemical assays wereperformed on hand cut sections as described in Jefferson (1987), supra.

At least ten events (transgenic plants) from each construct (pCGN2917and pCGN2926) were sent to the Growth Chambers/Greenhouse. Approximately80% (9/11) of the 2917 plants and 100% (12/12) of the 2926 plantsexpressed GUS at a level detectable by either fluorometric orhistochemical assay. Squares from several of pCGN2917 and pCGN2926transfected plants were assayed for GUS expression using histochemicalanalysis wherein the cells which are expressing GUS stain blue.Preliminary analysis indicates that all plants expressed GUS in thedeveloping floral parts. Ovules and anthers stained extremely dark.Bracts and locule walls were also blue in some cases. Fibers from 5, 9and 12 DPA bolls off these plants were also expressing GUS.

Several GUS assays were done on developing bolls at stages from squaringthrough 53 days post anthesis. GUS activity is very high in squares andflowers. Activity in bolls varies from plant to plant. Activity waspresent in fiber from two of the 2926 plants at 43 and 53 dpa.

β-glucuronidase is a very stable enzyme; therefore, presence of GUSactivity may not be directly correlated in a temporal manner with geneexpression, however, the specificity of expression in tissues and/orstructures derived from ovary integument was significant. Other tissuesnot derived from ovary integument, showed no GUS activity abovebackground. Differences in the breakdown of GUS as well as differencesin expression may explain the variability of expression patterns.

Comparisons between Cotton and Tomato Expression

An initial MUG assay was done on tissues from tomato and cotton plantstransfected with pCGN2917 and pCGN2918. GUS activity was found in tomatoroots, stems and leaves as well as meristems, and floral parts. Theamount of activity varied from plant to plant. In cotton, activity washighest in floral parts but was detectable in roots and stems of someplants.

T2 tomato plants from 2926 and 2917 are being tagged at anthesis. Theseplants have been tested for both kan and GUS expression. As the tissuematures it will be assayed and photographed.

EXAMPLE 10 Expression of Transaenic Melanin Synthesis Genes

A binary construct for plant transformation to express genes for melaninsynthesis is prepared as follows. The mel operon of Streptomycesantibioticus (Bernan et al. (1985) 34:101-110) is subcloned as a BclIfragment into a Bluescript vector. NcoI and BanHI sites are inserted bymutagenesis immediately 5′ to (and including) the ATG initiation codonfor ORF438. The resulting plasmid is pCGN4229. pCGN4229 is furthermutagenized by inserting a PstI site immediately following the ORF438stop codon and by the addition of NcoI and BamHI sites at the startcodon of the tyrA locus, thus, providing the mutagenized mel operon. APstI site from the plasmid vector is similarly located immediately 3′ tothe tyrA encoding region.

The pZ130 cassette, pCGN2909, is mutagenized to reinsert the NcoI siteincluding the ATG codon for the initial MET of the pZ130 encodedsequence, and results in pCGN4228. pCGN4228 is mutagenized to delete theBamHI site at the 3′ end of the pZ130 transcriptional termination regionand to insert an AscI linker fragment in its place, resulting inpCGN4235. pCGN4228 is also mutagenized to deleted the 3′ BamHI site andinsert an AscI linker 5′ to the pZ130 transcriptional initiation region(at XhoI/SalI digested and Klenow treated pCGN4228) resulting inpCGN4241.

The Streptomyces ORF438 region is obtained by digestion of themutagenized mel operon construct with NcoI and PstI and inserted intoNco/Pst digested pCGN4235. The tyrA region is cloned as an NcoI/PstIfragment from the mutagenized mel operon construct into Nco/Pst digestedpCGN4241.

A fragment of the tobacco ribulose bisphosphate carboxylase smallsubunit gene encoding the transit peptide and 12 amino acids of themature protein is inserted in reading frame with the ORF438 encodingsequence as an NcoI/BamHI fragment. The fragment is similarly insertedin front of the tyrA encoding sequence. The resulting constructs containthe transit peptide/ORF438 and transit peptide/tyrA fusions positionedfor expression from the pZ130 5′ and 3′ regulatory regions.

A binary vector (See FIG. 7) for insertion of the ORF438 and tyrAconstructs is prepared from pCGN1578 (McBride et al., supra) bysubstitution of the pCGN1578 linker region with a linker regioncontaining the following restriction digestionsites:Asp718/Asc/Pac/XbaI/BamHI/Swa/Sse/HindIII. (See FIG. 8). Thisresults in pCGN1578PASS. Asc, Pac, Swa and Sse are restrictive enzymesthat cut at the 8-base recognition sites. The enzymes are available fromNew England BioLabs: Asc, Pac; Boehringer Manheim:Swa; and Takara(Japan):Sse.

The ORF438 pZ130 construct is inserted into pCGN1578PASS as an Asp/Ascfragment. The tyrA pZ130 construct is inserted adjacent to the ORF438pZ130 construct as an Asc/Xba fragment.

EXAMPLE 11 Expression of Transaenic Melanin Synthesis Genes in TobaccoPlants

Transgenic tobacco plants were generated using techniques and DNAconstructs as provided in Examples 8-10.

A set of untransformed plants was utilized as a control. All of theuntransformed control plants utilized in this following experimentexhibited normal growth and development phenotypes. (See Table 1.)

A first set of transgenic plants was obtained using binary vectorpCGN4269 which expressed both the ORF438 and tyrA genes involved inmelanin synthesis in the cytosol of these tobacco plants. Transgenicplants obtained using pCGN4269 contained a DNA construct containing thepZ130 transcriptional and translational region from tomato which wasused to drive expression of the OFR438 and tyrA gene products.Cytosol-specific expression of the melanin synthesis genes yieldedtransgenic plants having a normal phenotype as compared to untransformedcontrol tobacco. (Table 1.) Melanin synthesis is not detectable in theseplants as the substrates for melanin production are not expected to bepresent at high levels in the cytosol.

A second set of transgenic plants was obtained using binary vectorpCGN4272 which specifically targeted the polypeptides expressed from themelanin synthesis genes to the plastids of these plants. Transgenicplants transformed with pCGN4272 contained a DNA construct containingthe tomato pZ130 transcriptional and translational initiation region andDNA encoding a tobacco SSU transit peptide and a 6 amino acid region ofthe mature SSU polypeptide coupled to the OFR438 gene and DNA encoding atobacco SSU transit peptide and a 6 amino acid region of the mature SSUpolypeptide coupled to the tyrA gene. The transit peptide was used todirect the transport of the ORF438 and tyrA gene products to theplastids of these plants. Plastid-targeted expression of the melaninsynthesis ORF438 and tyrA products resulted in plants having alteredphenotype (see Table 1). The phenotypic alterations included meristemabortion, stunted growth, narrow leaves, and new leaf curling.Alteration of plant color was also observed: some of the transgenicplants exhibited meristem yellowing and black streaks over variousportions of the plant and different meristimatic regions relative tocontrol plants. In addition, the basal flower buds of these transgenicplants were extremely dark compared to those transgenic plants whichexpressed the cytosol-specific melanin synthesis gene products orcompared to control plants. The pZ7 promoter is known to result inforeign gene expression in ovary and meristem derived tissue. Theobservation of this phenotype is believed to be due to depletion of thetyrosine amino acid pools in the plastid and/or the effect of auxin-likemelanin compounds on plant growth and development.

TABLE 1 Number of Plants Generated Plants Having Phenotype AlteredControl 20 0 Cytosol-Specific DNA 40 0 Construct Plastid-Specific DNA 5240 Construct

EXAMPLE 12 Constructs for Targeting Pigment Synthesis Genes

Constructs which contain encoding sequences for bacterial genes involvedin biosynthesis of pigmented compounds and sequences for directingtransport of the encoded proteins into plastids or vacuoles areprepared. The sequences are manipulated to be present on an NcoI/EcoRIfragment which may then be further manipulated to add transcriptionalinitiation regions useful for providing transcription in plant tissues.Examples of useful promoters include pZ7, T7 (for plastid expression),and various promoters capable of providing for expression in cottonfibers or plant flower petals.

For plastid targeting, the constructs contain a fragment of the tobaccoribulose bisphosphate carboxylase small subunit gene encoding thetransit peptide and 12 amino acids of the mature protein (Tssu)positioned in reading frame with the appropriate encoding sequence. Forproduction of indigo, pCGN5128 (Tssu::tna) and pCGN5129 (Tssu::pig) finduse for plastid targeting. The designation tna stands for the geneencoding tryptophanase from E. coli, an enzyme which converts tryptophanto indole (Stewart et al., (1986) J Bacteriol 166:217-223). The pigdesignation is used for the encoding sequence to the protein for indigoproduction from Rhodococcus, which produces indigo from indole (Hart eta (1990) J Gen Microbiol 136:1357-1363). Both tna and pig obtained byPCR. In pCGN5128 and pCGN5129 the transit SSU includes the tobacco 54amino acid transit peptide plus 12 amino acids from the mature smallsubunit protein.

For production of melanin in plants, constructs pCGN5075 (Tssu::TyrA)and pCGN5076 (Tssu::ORF438) find use for plastid targeting. In thisapproach melanin synthesis comes from the expression of two proteinsfrom Streptomyces antibioticus, the tyrA which converts tyrosine tomelanin and the ORF438, which is believed to assist the tyrA enzyme incopper binding (Bernan et al., (1985) Gene 37:101-110). Both proteinswere obtained by PCR. In pCGN5076 and pCGN5075 the transit from SSU alsoincludes the tobacco 54 amino acid transit peptide plus 12 amino acidsfrom the mature small subunit.

For vacuolar targeting of the melanin synthesis genes, constructsinclude a fragment of the metallocarboxypeptidase inhibitor gene,encoding the entire 32 amino acid N-terminus signal peptide of thatprotein plus 6 amino acids of the mature protein (CPI+6) (Martineau etal., supra), positioned in reading frame with the appropriate encodingsequences. In addition to the signal peptide, a sequence encoding avacuolar localization signal (VLS) is inserted 3′ of the proteinencoding sequence. Thus, for melanin production in vacuoles,CPI+6::tyrA::VLS and CPI+6::ORF438::VLS are used. In this example, theVLS utilized is the 8 amino acids obtained from beyond the C terminus ofthe metallocarboxypeptidase inhibitor gene described in Martineau et al.

As shown by the above results, expression of a gene of interest can beobtained in cells derived from ovary cells, including tomato fruit andcotton fibers, and expression of genes involved in synthesis of pigmentscombined with appropriate targeting sequences results in modification ofcolor phenotype in the selected plant tissue.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail, byway of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art that certain changes and modifications may be made thereto,without departing from the spirit or scope of the appended claims.

6 564 base pairs nucleic acid double linear cDNA to mRNA not provided 1AAA AAA ACA AAA ACA TTT CTA ATC TTT TTC ACT CAT TCC ATG GCT CGT 48 LysLys Thr Lys Thr Phe Leu Ile Phe Phe Thr His Ser Met Ala Arg 1 5 10 15TCC ATT TTC TTC ATG GCA TTT TTG GTC TTG GCA ATG ATG CTC TTT GTT 96 SerIle Phe Phe Met Ala Phe Leu Val Leu Ala Met Met Leu Phe Val 20 25 30 ACCTAT GAG GTA GAA GCT CAG CAA ATT TGC AAA GCA CCA AGC CAA ACT 144 Thr TyrGlu Val Glu Ala Gln Gln Ile Cys Lys Ala Pro Ser Gln Thr 35 40 45 TTC CCAGGA TTA TGT TTT ATG GAC TCA TCA TGT AGA AAA TAT TGT ATC 192 Phe Pro GlyLeu Cys Phe Met Asp Ser Ser Cys Arg Lys Tyr Cys Ile 50 55 60 AAA GAG AAATTT ACT GGT GGA CAT TGT AGC AAA CTC CAA AGG AAG TGT 240 Lys Glu Lys PheThr Gly Gly His Cys Ser Lys Leu Gln Arg Lys Cys 65 70 75 80 CTA TGC ACTAAG CCA TGT GTA TTT GAC AAA ATC TCA AGT GAA GTT AAA 288 Leu Cys Thr LysPro Cys Val Phe Asp Lys Ile Ser Ser Glu Val Lys 85 90 95 GCA ACT TTG GGTGAG GAA GCA AAA ACT CTA AGT GAA GTT GTG CTT GAA 336 Ala Thr Leu Gly GluGlu Ala Lys Thr Leu Ser Glu Val Val Leu Glu 100 105 110 GAA GAG ATT ATGATG GAG TAATAA TTA AGT GAG GTT AAA TAA GGA TTT 384 Glu Glu Ile Met MetGlu Leu Ser Glu Val Lys Gly Phe 115 120 125 TGA GTG TCA AAA AAA ACA AAATTA ATA AAG TGT TGC CTT TTC TTA TTA 432 Val Ser Lys Lys Thr Lys Leu IleLys Cys Cys Leu Phe Leu Leu 130 135 140 GGG TAG CTT GTG ATG TTG TGT TAGTAT TGG CCT ATA GTA GCC ATT TGA 480 Gly Leu Val Met Leu Cys Tyr Trp ProIle Val Ala Ile 145 150 CAC ATT AAA TAA GTT TGT GAC ACA TCA TTA ATC CTTATG TAT GTA TGT 528 His Ile Lys Val Cys Asp Thr Ser Leu Ile Leu Met TyrVal Cys 155 160 165 TTT AAT GAA AAA TGA TCG ACT ACG ATC TTT AAT TTT 564Phe Asn Glu Lys Ser Thr Thr Ile Phe Asn Phe 170 175 3528 base pairsnucleic acid double linear genomic DNA not provided 2 GCTCCACTACTCTCATCACT TTAGTTCATC AAGCCTTCTT TTATACCAAG GCATCATCAA 60 TCTCATTAACAAAGTAGATT AGGGTTTTTC AAGATTTAGG ATTCAATAGC TTCATCATGC 120 TTATTTTATCACAATTATAT AATCACATTC ATACAAGCAT ACAATTAAGC ATATAGAAGG 180 GTTTACAATACTACCCAATA CATATCATTC GCTATTAAGA GTTTACTACG AATAGCATAA 240 ACCATAACCTACCTCCACCG AAGAATCGCG ATCAAACAAT CTACTTTCCC AAAGCTGCGT 300 TCTTCTTCGTTTTCTCTCTC TCTTGATCGT TCGTTTCTCC CTCTCTTTGT TCTTTCTATT 360 TTTCTTATTCAAACCCTCTT TCTTTTACCC TAATTAGTAT ATAATTAAGT ATAAAAGATG 420 ATAAAATACCCCATCTATTT GTTTGAAGGT TATCTCTTTT AGCCCCCAAG TAATTGAATT 480 ATTAACATTAAACCACTAAC TTTATAATTA TAAGCAGGAA TAGTCCAAAA CGCCCCTTAA 540 AATATTTAACAGAAATCCGA CCCAGTCAGG GTCACGCAGC CTGTANCGGN NCACAACTGT 600 GACGGTCCGTCCTGCATGGC CGTCACAAAG TTCAGAGAGT TAATTTCTGT GGAAGATGTG 660 TANGGTNGTCGTGCCCACGA CGGTCCGTCC TGTCATTTCG TTACGAAGTT CAGAGAGTCG 720 ATTTCAGTACCCAAATTTCA GAATTCTAAG TGTTTTGGAA CGAGACCCCN CGGTCCGTCG 780 TGCCCATGACGGTTCGTCGT GGGATCCGTC GACTCAGCCA GTTTTTCCAA AATTAAAATC 840 TGCTGCTCAAAACGACTAAA CAGGTCGTTA CAAAGTACTC AATCAAATAA AAAGAATAAA 900 TTCTTTTCCAAATACATATA TTGTTTATAG GACAGTGTTA ACAGGGAAAT GTAATCGTTG 960 CCTCAATCGATTTTTTTTTT TGAAATTAAG ATTGATTAGA TCTTCTTTAA GATAACAATG 1020 TCTCAAAGATAAATTGAATG AATGAATTAG CTATATTATC ATTTGAAAAG AAATTACTAA 1080 AACAGATTGATAATAAAATA ATAATAAATG ACTTTGCATC TAAAATAGCT AGAAAGCAGA 1140 TTTTTAAATAAAAATACATA TGATAAAAAA AAGATAAATT AGAGTCATCC CATAAATTTC 1200 GCTTTAGGCCCCCAATGTTG TTAAGTCGGC CCTGAAAATA GGAATGGTAT TAAATATTTT 1260 GTTTTGATTTCACACTTGAT ATTTGACATT CATATTAGAA AATAATTAAA TTTATATTCG 1320 TGTAGAGTGGTCTCACATTA ATGGGTAAAA TATTTCCACA CAAAAACTAT TTTACAATCA 1380 TAGCTAGAATCTGAAATATC TAATGTACTC CACCCAATTA ATTAAAGATG ATTTTTTTGC 1440 TTAAATAATAAAAATATGTC TATTGCCAAA CTACTAATAG ATGTACTCAC AAAAAAAATA 1500 AAATAAAAAATCAAGTGTAT ATACAATGAT TCGGAAGGCC ATTTTTGAAA ATTTTCATAA 1560 AATGACCGTTTTACCCGTTC ACAATTGTTG TTTCAGCATT TTTGTTTGGT TTGTGGATTT 1620 GGTTATGGAAGTTCAATAAA AAGTTGTGGT TTTATAAGCT TTGGAGTTTT GAAAGGTTTA 1680 AGTTGATTAAAAGTAGTTTT TAGTGTCAAT TGGAGTTTCG TGTCTTGAAA TAAATTTTAT 1740 CACTTGCATTAGTTTCAAAA TGTCGAGTTT GGTTAAGTAG AGGTTTTTTT CATTCGGAGT 1800 TTTTTTATGAATTTAAAATG TTAAGCTGAA AGTTTATGAA ATTTTAGCCT TTGAGTTAAT 1860 TTTGATGCTTGAATTAAATT TTTGAGAATT TTTTTGAAAT CTGGGGATAA TGTTAGGTCT 1920 TAGAGAAGTCTGGTTGAATT TTCATAGCTC AAGAGATTAG TTTTGACTTT TTAGGCATTT 1980 TGTTGGTTTATTACGATTTT CACGGACTTT CGAATTAAGG AGACTTCAAA ATTCATATTT 2040 AATGGTTCGTGTGTTCGTTA GTTTTAAAAA TCGTGTCTTT ATAAGGATTT ATACTTAAAA 2100 AAATAAAATAAAATAAAGTA CTACTAACAT GTAATTCTGT CATAAGATAA GGTTGTACAT 2160 TTAGGACTATTTGAATATTC ATCAAAAATA AAAAAAAGTA GAGATGATAG TAATATAAAT 2220 ATTTATTTTTGATTTTACAT TTGATATTTT AATACTAACA ATATGACATA ATAAAATTTG 2280 TATTCAGATTGTAAAATATT CCCTAAAAAA AGATACTTTT ACTGTGGTGG CTCAAATTCA 2340 AAATTTTCTAAGAAAAACTA CTAATAATTG ATTTCTAATT AAAATTTCGA TATATATATA 2400 TATATATATATATATATCAT AATATACTTC ACCTACCTCA ATTATTATTA TTTTCTTTTT 2460 TTTTTACTTCACATATTTTT GGSCSACCAA TTTTTTTTTT AACTTTTTTG GTCTTACTCT 2520 TATTTCACTCCCTATAAATA ACTCCCATTG TGTGATATTT TTATTCACAA CTCTAACTTA 2580 CAATCTTTCTTATTATTAAA AAAAACAAAA ACATTTCTAA TCTTTTTCAC TCATTCCATG 2640 GCTCGTTCCATTTTCTTCAT GGCATTTTTG GTCTTGGCAA TGATGCTCTT TGTTACCTAT 2700 GGTTTGTCTTCATAATTTAT TCCTCTAAAA TCATCGCAAT AAAAAAAAAA TGTAACGAAG 2760 CAGACATCAGTAAACCGTTT AAATAAACCC TAAAAAAATT GTGAATTGAT ATTACTTGCT 2820 ATACGTTTAACAACTATGAT AAAAAAACCC TAAAATATAC TTATTTCGAT TTCGTCTCTC 2880 TCATGTTATTCTAACTATTT TTTGTGTGTG AATGATTGTA GAGGTAGAAG CTCAGCAAAT 2940 TTGCAAAGCACCAAGCCAAA CTTTCCCAGG ATTATGTTTT ATGGACTCAT CATGTAGAAA 3000 ATATTGTATCAAAGAGAAAT TTACTGGTGG ACATTGTAGC AAACTCCAAA GGAAGTGTCT 3060 ATGCACTAAGCCATGTGTAT TTGACAAAAT CTCAAGTGAA GTTAAAGCAA CTTTGGGTGA 3120 GGAAGCAAAAACTCTAAGTG AAGTTGTGCT TGAAGAAGAG ATTATGATGG AGTAATAATT 3180 AAGTGAGGTTAAATAAGGAT TTTGAGTGTC AAAAAAAACA AAATTAATAA AGTGTTGCCT 3240 TTTCTTATTAGGGTAGCTTG TGATGTTGTG TTAGTATTGG CCTATAGTAG CCATTTGACA 3300 CATTAAATAAGTTTGTGACA CATCATTAAT CCTTATGTAT GTATGTTTTA ATGAAAAATG 3360 ATCGACTACGATCTTTAATT TTATGTTTTA CATTTAATTA ATCACTTTCT GTTACGATTC 3420 ATTTATCTAGTTATGAATGA AATATAGAGT GATTTGAAGT AAGGAGCTAG TCTTCAAACA 3480 AAGACGTACATATGTACAAA GTAGGGTACT ATTAAACTTC TTTTTTAT 3528 453 base pairs nucleicacid double linear cDNA to mRNA not provided 3 ATTATTATTA CC ATG GCA CAAAAA TTT ACT ATC CTT TTC ACC ATT CTC CTT 51 Met Ala Gln Lys Phe Thr IleLeu Phe Thr Ile Leu Leu 1 5 10 GTG GTT ATT GCT GCT CAA GAT GTG ATG GCACAA GAT GCA ACT CTG ACG 99 Val Val Ile Ala Ala Gln Asp Val Met Ala GlnAsp Ala Thr Leu Thr 15 20 25 AAA CTT TTT CAG CAA TAT GAT CCA GTT TGT CACAAA CCT TGC TCA ACA 147 Lys Leu Phe Gln Gln Tyr Asp Pro Val Cys His LysPro Cys Ser Thr 30 35 40 45 CAA GAC GAT TGT TCT GGT GGT ACG TTC TGT CAGGCC TGT TGG AGG TTC 195 Gln Asp Asp Cys Ser Gly Gly Thr Phe Cys Gln AlaCys Trp Arg Phe 50 55 60 GCG GGG ACA TGT GGG CCC TAT GTT GGG CGC GCC ATGGCC ATA GGC GTG 243 Ala Gly Thr Cys Gly Pro Tyr Val Gly Arg Ala Met AlaIle Gly Val 65 70 75 TGATTACAAT TTCGTTGTTC TTCTTTTTCG ACTTTTTAATCCCAAGTGAA TAAAGTCTAA 303 TTCGAAAAAG AAGAAAAAAG TATCTATGTC TGAGTTATATGTTTTGTGGC TAATAAGAAA 363 TCGACTATGC TTGTTGATTT GATAAAAATT ATGTCATTAGGGTGTGATAT GTAATCATCA 423 AATTAAATAA AAATCATCGC ATTGTGTGTG 453 36 basepairs nucleic acid single linear other nucleic acid syntheticoligonucleotide not provided 4 AATTAGATGC AGGTCCATAA GTTTTTTCTA GACGCG36 42 base pairs nucleic acid single linear other nucleic acid syntheticoligonucleotide not provided 5 GTTCCTGCAG CATGCCCGGG ATCGATAATAATTAAGTGAG GC 42 25 base pairs nucleic acid single linear other nucleicacid synthetic oligonucleotide not provided 6 CAAGAATTCA TAATATTATATATAC 25

What is claimed is:
 1. A DNA construct comprising as operably joinedcomponents in the direction of transcription, a tomato pZ7 promoter, atransport signal encoding sequence from a plant nuclear-encoded gene,and an open reading frame encoding an enzyme required for synthesis of apigment.
 2. A plant cell comprising the DNA construct of claim
 1. 3. Aplant comprising the cell of claim
 2. 4. The DNA construct according toclaim 1 wherein said transport signal encoding sequence encodes aplastid transit peptide.
 5. The DNA construct according to claim 4,wherein said transport signal encoding sequence further comprises aportion of the mature protein encoding region for said plantnuclear-encoded gene.
 6. The DNA construct according to claim 1 whereinsaid transport signal encoding sequence encodes a signal peptide whichprovides for transport across the rough endoplasmic reticulum.
 7. TheDNA construct according to claim 6, wherein said DNA sequence furthercomprises, 3′ to said open reading frame, a vacuolar localizationsignal.
 8. The DNA construct of claim 1 wherein said pigment is melaninor indigo.
 9. The DNA construct of claim 8 wherein said open readingframe is from a bacterial gene.
 10. The DNA construct of claim 9 whereinsaid bacterial gene is selected from the group consisting of ORF438,tyrA, pig, and tna.
 11. A plant cell comprising the DNA constructaccording to anyone of claims 4-10.
 12. A plant comprising a cell ofclaim
 11. 13. A method of modifying color phenotype in a cotton fibertissue, said method comprising: transforming a plant cell with DNAcomprising a construct for expression of a protein in a melaninbiosynthesis pathway, wherein said construct comprises as operablyjoined components: i) a promoter for transcription in a cotton fibercell, ii) a transport signal encoding a plastid transit peptide, iii) atyrA or an ORF438 open reading frame, and iv) a transcriptionaltermination region functional in cells of said cotton fiber tissue,wherein said cotton fiber tissue comprises a substrate of a proteinencoded by tyrA or ORF438; and growing said plant cell to produce aplant comprising said cotton fiber tissue, wherein said protein reactswith said substrate to produce melanin.
 14. A method of modifying colorphenotype in a cotton fiber tissue, said method comprising: transforminga plant cell with DNA comprising constructs for expression of twoproteins in a melanin biosynthesis pathway, wherein each constructcomprises as operably joined components: i) a promoter for transcriptionin a cotton fiber cell, ii) a transport signal encoding a signal peptidewhich provides for transport across the rough endoplasmic reticulum,iii) tyrA and ORF438 open reading frames, and iv) a transcriptionaltermination region functional in cells of said cotton fiber tissue,wherein said cotton fiber tissue comprises substrate of proteins encodedby tyrA and ORF438; and growing said plant cell to produce a plantcomprising said cotton fiber tissue, wherein said proteins react withsaid substrate to produce melanin.
 15. A method of modifying colorphenotype in a cotton fiber tissue, said method comprising: transforminga plant cell with DNA comprising a construct for expression of a proteinin a pigment biosynthesis pathway, wherein said construct comprises asoperably joined components: i) a tomato pZ7 promoter, ii) a transportsignal encoding sequence from a plant nuclear-encoded gene, iii) an openreading frame encoding a protein required for synthesis of a pigment,and iv) a transcriptional termination region functional in cells of saidcotton fiber tissue, wherein said cotton fiber tissue comprises asubstrate of said protein; and growing said plant cell to produce aplant comprising said cotton fiber tissue, wherein said protein reactswith said substrate to produce said pigment.
 16. The method of claim 15wherein said transport signal encoding sequence encodes a plastidtransit peptide.
 17. The method of claim 16 wherein said DNA comprisesconstructs for expression of two proteins in a pigment biosynthesispathway, wherein each of said constructs comprises components i) throughiv), and wherein said two proteins are not encoded by the same gene. 18.The method of claim 15 wherein said transport signal encoding sequenceencodes a signal peptide which provides for transport across the roughendoplasmic reticulum.
 19. The method of claim 18 wherein said DNAcomprises constructs for expression of two proteins in a pigmentbiosynthesis pathway, wherein each of said constructs comprisescomponents i) through iv), and wherein said two proteins are not encodedby the same gene.
 20. The method of claim 17 or 19 wherein said pigmentis melanin and said proteins are encoded by tyrA and ORF438.
 21. Amethod of modifying color phenotype in a cotton fiber tissue, saidmethod comprising: transforming a plant cell with DNA comprisingconstructs for expression of two proteins in a melanin biosynthesispathway, wherein each construct comprises as operably joined components:i) an ovary tissue promoter that directs transcription prior to anthesisthrough flower senescence in a cell that will develop into a cottonfiber cell, ii) a transport signal encoding a signal peptide whichprovides for transport across the rough endoplasmic reticulum, iii) tyrAand ORF438 open reading frames, and iv) a transcriptional terminationregion functional in cells of said cotton fiber tissue, wherein saidcotton fiber tissue comprises substrate of proteins encoded by tyrA andORF438; and growing said plant cell to produce a plant comprising saidcotton fiber tissue, wherein said proteins react with said substrate toproduce melanin.
 22. A method of modifying color phenotype in a cottonfiber tissue, said method comprising: transforming a plant cell with DNAcomprising a construct for expression of a protein in a melaninbiosynthesis pathway, wherein said construct comprises as operablyjoined components: i) an ovary tissue promoter that directstranscription in a cell that will develop into a cotton fiber cell priorto anthesis through flower senescence, ii) a transport signal encoding aplastid transit peptide, iii) a tyrA or an ORF438 open reading fame, andiv) a transcriptional termination region functional in cells of saidcotton fiber tissue, wherein said cotton fiber tissue comprises asubstrate of a protein encoded by tyrA or ORF438; and growing said plantcell to produce a plant comprising said cotton fiber tissue, whereinsaid protein reacts with said substrate to produce melanin.